The invention relates to ancrod-specific monoclonal antibodies, antibody fragments, mixtures or derivatives thereof and their use in pharmaceutical preparations or in diagnosis, and to pharmaceutical preparations which comprise these antibodies, antibody fragments, mixtures or derivatives thereof.
The invention furthermore relates to cells which express these antibodies, antibody fragments, mixtures or derivatives thereof.
Ancrod (proprietary name: Arwin(copyright), Arvin(copyright)) is an enzyme from the venom of the Malayan pit viper (Agkistrodon rhodostoma). It is a highly glycosylated serine protease which has an average MW of about 38000 and which has anticoagulant properties and the ability to dissolve blood clots.
Normal coagulation of blood is effected by thrombin which eliminates fibrinopeptides A and B from the fibrinogen molecule and thus leads to the formation of fibrin (EP-B-0 556 906), the main constituent of thrombi in addition to, for example, red blood corpuscles or platelets. In contrast to thrombin, acrod cleaves only the arginine-glycine linkage in the a(xe2x80x9cAxe2x80x9d) chain of the fibrinogen molecule, which liberates fibrinopeptides A, AP and AY (Cole et al., J. Vascular. Surgery, Vol 17, 1993: 288-292). The xcex2(B) chain of the fibrinogen molecule is not attacked by ancrod and is thus not liberated. The fragments (de-xe2x80x9cAxe2x80x9d-fibrin monomers) produced after the elimination of the fibrinopeptides caused by ancrod are eventually able to polymerize to thin filaments. The resulting atypical, soluble fibrin is lyzed by endogenous plasmin and/or removed by the reticuloendothelial system (=RES, monocyte/macrophage system). Further cleavage of the de-xe2x80x9cAxe2x80x9d-fibrinogen molecule by thrombin to give natural fibrin no longer takes place because the resulting molecule is not a thrombin substrate.
Ancrod causes a dose-dependent decrease in the blood fibrinogen concentration. Therapeutically induced and controlled hypofibrinogenemia diminishes the plasma viscosity and tendency of erythrocytes to aggregate so far that the flow properties of the blood are crucially improved. This provides the condition for greater flow of blood through stenosed vessels. Ancrod is currently used to treat, for example, chronic disturbances of peripheral arterial blood flow, and is undergoing clinical phase III studies on stroke.
Ancrod is advantageously injected subcutaneously. Treatment can take place in hospital or, if the regular checks of the fibrinogen concentration necessary to monitor the therapy are ensured, also on an outpatient basis. Intravenous administration of ancrod is possible but should take place only in exceptional cases and under hospital observation.
The dosage of ancrod must also be individualized. The behavior of the fibrinogen concentration as a function of the ancrod dose is crucial. It must be slowly reduced to 70-100 mg/100 ml of plasma (=therapeutic range). The fibrinogen concentration must be adjusted to be within this range throughout the treatment period. The flow properties of the blood are satisfactory under these conditions. The therapy normally lasts 3-4 weeks but can, if necessary, be extended beyond this period.
On subcutaneous administration, 70 I.U. (=international units, 1 ml) are given each day in the first 4 days, and 70-140 I.U. are given, depending on the behavior of the fibrinogen concentration, from day 5 onwards. If the fibrinogen concentration is in the therapeutic range, single injections of 210-280 I.U. are given 2-3 times a week.
On intravenous infusion, initially 2-3 I.U./kg of body weight are given over the course of eight hours. The subsequent dosage of ancrod depends on the fibrinogen concentration attained. It is generally sufficient to inject a further 1 I.U./kg of body weight slowly every 12 hours.
The initial half-life of ancrod in the circulation is about 3-5 hours, but slows down as the concentration falls so that after about 4 days, within this time in general 90% of the administered ancrod are eliminated, the half-life is extended to 9-12 days.
Although ancrod contrasts with, for example, heparin and warfarin in being associated with fewer problems of unspecific bleeding during the treatment (see Z. S. Latallo, xe2x80x9cRetrospective Study on Complications and Adverse Effects of Treatment with Thrombin-Like Enzymesxe2x80x94A Multicenter Trialxe2x80x9d, Thromb. Haemostasis, 50 (1983) 604-609), specific treatment of such bleeding is necessary and desirable.
Contraindications for treatment with ancrod are, for example, hemorrhagic diathesis, danger of bleeding associated with injuries, after operations and deliveries, for ulcerative intestinal disorders, neoplasms, poorly controllable hypertension, acute cerebral infarct and active pulmonary tuberculosis, dysfunctions of the RES and disturbances of clot breakdown, eg. in states of high fever, severe liver disorders, manifest and incipient states of shock or pregnancy.
As described above, the risk of bleeding is relatively low with ancrod when the fibrinogen concentration is reduced slowly and is adjusted to 70-100 mg/100 ml during the period of therapy. Patients with a latent tendency to bleed, eg. cases of kidney stones or renal failure, should be monitored particularly carefully. Arterial punctures and intramuscular injections of other drugs should be avoided. Caution is necessary on concurrent administration of RES-blocking and ulcerogenic drugs, anticoagulants, antifibrinolytics, thrombolytics and medicines which inhibit platelet aggregation, and on intramuscular administration of ancrod. Absorption from the muscle depot generally takes place very quickly so that too many de-xe2x80x9cAxe2x80x9d-fibrin monomers flow away and there is a danger of thromboembolic complications.
The total incidence of bleeding in a study on 429 patients (Crit. Rev. Oncol. Hematol. 15 (1993) 23-33), who received ancrod without previous thrombolytic therapy was 9.8% (4.2% internal bleeding; 5.6% external bleeding).
Currently used to neutralize the enzymatic activity of ancrod is an antidote based on an immunoglobulin preparation from goat serum (Knoll AG publication, June 1983, entitled Arwin(copyright)). This antidote consisting of polyclonal antibodies is used in cases of severe hemorrhagic complications or increased danger of bleeding, eg. associated with accident injuries or because surgery suddenly becomes indicated. Neutralization of ancrod should be followed by administration of 4-5 g of human fibrinogen. If human fibrinogen, plasma or blood is administered without previous neutralization of ancrod by an antidote, there is a danger of acute disseminated coagulation.
Stocker et al (Thrombosis Research, Vol. 6, 1975: 189-194) investigated thrombogenesis in the presence of Arwin(copyright) alone and in the presence of the polyclonal antidote and were able to demonstrate the antidote effect.
Besides this use of polyclonal antibodies from goats, EP-B-0 395 375, EP-B-0 556 906 and Burkhardt et al (FEBS, Vol 297, No. 3, 1992: 297-301) describe monoclonal or polyclonal antibodies for detecting expression of ancrod genes, for detecting fibrinogen in blood using ancrod and ancrod antibodies or purification of ancrod using antibodies.
A disadvantage of the goat polyclonal antibodies used as ancrod antidote is that, for example, they consist of a mixture of antibodies, many of which have no ancrod-neutralizing effect. This large number of different antibodies may lead to a rapid immune response and, moreover, leads to a relatively low ancrod-neutralizing capacity. In addition, the antidote contains antibodies of varying affinity for ancrod. Polyclonal antibodies can, because they are obtained from animals, be standardized only with difficulty, which means that there are variations in the different production batches.
It is an object of the present invention to develop an antidote to ancrod which does not have the abovementioned disadvantages and is easy to produce industrially.
We have found that this object is achieved by the novel monocloncal antibodies, antibody fragments, mixtures or derivatives thereof which bind to ancrod and inhibit its activity, where the binding affinity is in a range from 1xc3x9710xe2x88x927 to 1xc3x9710xe2x88x9212 M, and the neutralizing effect is improved at least 100% by comparison with goat polyclonal antibodies in vivo.